Controlling Intracellular Machinery via Polymer Pen Lithography Molecular Patterning

The plasma membrane and the actomyosin cytoskeleton play key roles in controlling how cells sense and interact with their surrounding environment. Myosin, a force-generating actin network-associated protein, is a major regulator of plasma membrane tension, which helps control endocytosis. Despite the important link between plasma membranes and actomyosin (the actin–myosin complex), little is known about how the actomyosin arrangement regulates endocytosis. Here, nanoscopic ligand arrangements defined by polymer pen lithography (PPL) are used to control actomyosin contractility and examine cell uptake. Confocal microscopy, atomic force microscopy, and flow cytometry suggest that the cytoskeletal tension imposed by the nanoscopic ligand arrangement can actively regulate cellular uptake through clathrin- and caveolin-mediated pathways. Specifically, ligand arrangements that increase cytoskeletal tension tend to reduce the cellular uptakes of cholera toxin (CTX) and spherical nucleic acids (SNAs) by regulating endocytic budding and limiting the formation of clathrin- and caveolae-coated pits. Collectively, this work demonstrates how the cell endocytic fate is regulated by actomyosin mechanical forces, which can be tuned by subcellular cues defined by PPL.


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Deposition of fibronectin on MHA patterns. Unetched glass substrates were patterned with MHA ink (Patterning MHA features). The substrates were backfilled with 1 mM solution of (1mercapto-11-undecyl)hexa(ethylene glycol) in ethanol for 1 hr to prevent non-specific binding of cells or proteins. Prior to incubation of fibronectin solution, the substrates were thoroughly rinsed with ethanol and H 2 O. A six-well plate was then filled with 50 µg/mL human plasma fibronectin (Millipore) in 1× phosphate buffered saline (PBS; pH 7.0) and placed on a shaker stirring (100 r.p.m.) overnight at 4 °C. The sample was then rinsed with 1× PBS after fibronectin incubation.

Seeding of NIH3T3s.
NIH3T3s were seeded on the patterned substrates that varied in their numbers of peripheral features at a density of 3,000 cells/cm 2 . Fibroblasts cells were cultured on patterned substrates overnight.
Immunofluorescence. After overnight incubation, the NIH3T3 cells on patterned substrates were fixed with 4% formaldehyde for 10 min and subsequently permeabilized with 0.1% Triton X-100 for 15 min followed by three rounds of washing. They were then blocked with 1% BSA diluted in 1× PBS for 1 hr. The substrates were then labeled with rabbit anti-myosin IIa (1:200, rabbit polyclonal to non-muscle Myosin IIA, Abcam ab75590) and mouse anti-vinculin (1:200, mouse monoclonal to Vinculin, Abcam ab130007) followed by secondary antibody staining (1:1000, goat S5 anti-rabbit Alexa Fluor 488, Invitrogen A32731. 1:1000, goat anti-mouse Alexa Fluor 555). The substrates were then mounted on a coverslip using Prolong Gold Antifade mountant with DAPI (Invitrogen) for visual observation using immunofluorescence microscopy.
In uptake experiments and qualitative studies of different endosomal pathways, cells were incubated with fluorescently labeled cholera toxin (CTX) conjugates for 1 hr. After washing the substrates with 1× PBS, the surfaces of the substrates were fixed with 4% formaldehyde and incubated with blocking solution (0.1% Triton X-100 and 1% BSA diluted in 1× PBS) for 1 hr.
The fixed cells then were labeled with rabbit anti-caveolin (1:200, rabbit polyclonal to caveolin-1, Abcam ab2910) and mouse anti-clathrin (1:200, mouse monoclonal to clathrin heavy chain, Abcam ab2731) diluted in 1× PBS overnight at 4 o C on a shaker. Secondary antibody labeling was performed using either Alexa-488 or Alexa-555 goat anti-rabbit or anti-mouse (Invitrogen). The substrates were further mounted onto the coverslips using the same mounting procedure as described above. Immunofluorescence confocal microscopy was performed using a Leica SP8.
Focal adhesion kinase phosphorylation assay. Prior to performing focal adhesion kinase phosphorylation assays, cells were trypsinized, neutralized with cell culture media and centrifuged at 500 x g for 5 min at 4 o C. Cell pellets were incubated with cold 1× cell extraction buffer on ice for 20 min followed by centrifugation at 18,000 x g for 20 mins at 4 o C. The supernatants were collected and transferred into clean tubes, and the pellets were discarded. The total protein concentration in the extract was quantified using a Pierce BCA protein assay kit. The phosphorylation of FAK at Y397 was assessed using an ELISA assay (Invitrogen) performed according to the manufacturer's protocol.   A B S14 Figure S5. The fluorescence signal of pHrodo green dextran specifically targeting endosomes was also slightly higher in cells seeded on the 35-point circle than on other shapes. Statistical analysis was performed using one-way ANOVA followed by multiple comparison tests using Tukey post hoc analysis. *: p < 0.05.